Moon L. Shin

Role of Tumor Necrosis Factor-α in Neuronal and Glial Apoptosis after Spinal Cord Injury

We investigated the role of tumor necrosis factor (TNF)-alpha in the onset of neuronal and glial apoptosis after traumatic spinal cord crush injury in rats. A few TUNEL-positive cells were first observed within and surrounding the lesion area 4 h after injury, with the largest number observed 24-48 h after injury. Double-labeling of cells using cell type-specific markers revealed that TUNEL-positive cells were either neurons or oligodendrocytes. One hour after injury, an intense immunoreactivity to TNF-alpha was observed in neurons and glial cells in the lesion area, but also seen in cells several mm from the lesion site rostrally and caudally. The level of nitric oxide (NO) also significantly increased in the spinal cord 4 h after injury. The injection of a neutralizing antibody against TNF-alpha into the lesion site several min after injury significantly reduced both the level of NO observed 4 h thereafter as well as the number of apoptotic cells observed 24 h after spinal cord trauma. An inhibitor of nitric oxide synthase (NOS), N(G)-monomethyl-l-arginine acetate (l-NMMA), also reduced the number of apoptotic cells. This reduction of apoptotic cells was associated with a decrease in DNA laddering on agarose gel electrophoresis. These results suggest that: (i) TNF-alpha may function as an external signal initiating apoptosis in neurons and oligodendrocytes after spinal cord injury; and (ii) TNF-alpha-initiated apoptosis may be mediated in part by NO as produced by a NOS expressed in response to TNF-alpha.

Role of the C5b-9 complement complex in cell cycle and apoptosis

Assembly of C5b‐9 on cell membranes results in transmembrane channels and causes cell death. When the number of C5b‐9 molecules is limited, nucleated cells are able to escape cell death by endocytosis and by shedding of membranes bearing C5b‐9. Sublytic C5b‐9 induces proto‐oncogenes, activates the cell cycle, and enhances cell survival. In addition, C5b‐9 reverses the differentiated phenotype of post‐mitotic cells, such as oligodendrocytes and skeletal muscle cells. The signal transduction pathways responsible for cell cycle activation by C5b‐9 include Gi‐mediated activation of extracellular signal‐regulated kinase 1 and phosphatidylinositol 3‐kinase (PI3‐K). Cell survival enhanced by C5b‐9 is mediated by the PI3‐K/Akt pathway, which inhibits apoptosis through regulation of BAD. These findings indicate that complement activation and membrane assembly of sublytic C5b‐9 play an important role in inflammation by promoting cell proliferation and by rescuing apoptotic cells.

C5b-9 Terminal Complement Complex Protects Oligodendrocytes from Death by Regulating Bad Through Phosphatidylinositol 3-Kinase/Akt Pathway

Apoptosis of oligodendrocytes is induced by serum growth factor deprivation. We showed that oligodendrocytes and progenitor cells respond to serum withdrawal by a rapid decline of Bcl-2 mRNA expression and caspase-3-dependent apoptotic death. Sublytic assembly of membrane-inserted terminal complement complexes consisting of C5b, C6, C7, C8, and C9 proteins (C5b-9) inhibits caspase-3 activation and apoptotic death of oligodendrocytes. In this study, we examined an involvement of the mitochondria in oligodendrocyte apoptosis and the role of C5b-9 on this process. Decreased phosphatidylinositol 3-kinase and Akt activities occurred in association with cytochrome c release and caspase-9 activation when cells were placed in defined medium. C5b-9 inhibited the mitochondrial pathway of apoptosis in oligodendrocytes, as shown by decreased cytochrome c release and inhibition of caspase-9 activation. Phosphatidylinositol 3-phosphate kinase and Akt activities were also induced by C5b-9, and the phosphatidylinositol 3-phosphate kinase inhibitor LY294002 reversed the protective effect of C5b-9. Phosphatidylinositol 3-phosphate kinase activity was also responsible for the phosphorylation of Bad at Ser112 and Ser136. This phosphorylation resulted in dissociation of Bad from the Bad/Bcl-xL complex in a Giα-dependent manner. The mitochondrial pathway of oligodendrocyte apoptosis is, therefore, inhibited by C5b-9 through post-translational regulation of Bad. This mechanism may be involved in the promotion of oligodendrocyte survival in inflammatory demyelinating disorders affecting the CNS.

Molecular Cloning and Characterization of RGC-32, a Novel Gene Induced by Complement Activation in Oligodendrocytes

Sublytic complement activation on oligodendrocytes (OLG) down-regulates expression of myelin genes and induces cell cycle in culture. Differential display (DD) was used to search for new genes whose expression is altered in response to complement and that may be involved in cell cycle activation. DD bands showing either increased or decreased mRNA expression in response to complement were identified and designated ResponseGenes to Complement (RGC) 1–32.RGC-1 is identical with heat shock protein 105,RGC-2 with poly(ADP-ribose) polymerase, andRGC-10 with IP-10. A new gene, RGC-32, that encodes a protein of 137 amino acids was cloned. RGC-32 has no homology with other known proteins, and contains no motif that would indicate its function. In OLG, the mRNA expression was increased by complement activation and by terminal complement complex assembly. RGC-32 protein was localized in the cytoplasm and co-immunoprecipitated with cdc2 kinase. Overexpression of RGC-32 increased DNA synthesis in OLGxC6 glioma cell hybrids. These results suggest thatRGC-32 may play a role in cell cycle activation.

C5b-9-induced endothelial cell proliferation and migration are dependent on Akt inactivation of forkhead transcription factor FOXO1.

Migration and proliferation of aortic endothelial cells (AEC) are critical processes involved in angiogenesis, atherosclerosis, and postangioplasty restenosis. Activation of complement and assembly of the C5b-9 complement complex have been implicated in the pre-lesional stage of atherogenesis and progression of the atherosclerotic lesion. We have shown that C5b-9 induces proliferation and activates phosphatidylinositol 3-kinase (PI3K), but it is unknown whether this can lead to activation of Akt in AEC, a major downstream target of PI3K, or if C5b-9 can induce the migration of AEC, a critical step in angiogenesis. In this study, we show that C5b-9 induces AEC proliferation and migration and also activates the PI3K/Akt pathway. C5b-9 activates Akt as shown by in vitro kinase assay and phosphorylation of Ser-473. C5b-9-induced cell cycle activation was inhibited by pretreatment with LY294002 (PI3K inhibitor), SH-5 (Akt inhibitor), or transfection with Akt siRNA. These data suggests that the PI3K/Akt pathway is required for C5b-9-induced cell cycle activation. FOXO1, a member of forkhead transcription factor family, was phosphorylated at Ser-256 and inactivated after C5b-9 stimulation as shown by a decrease in DNA binding and cytoplasmic relocalization. Cytoplasmic relocalization was significantly reduced after pretreatment with LY294002, SH-5, or transfection with Akt siRNA. Silencing FOXO1 expression using siRNA stimulated AEC proliferation and regulated angiogenic factor release. Our data indicate that C5b-9 regulation of the cell cycle activation in AEC through Akt pathway is dependent on inactivation of FOXO1.

The Glomerular Complement Receptor in Immunologically Mediated Renal Glomerular Injury

We examined 25 renal-biopsy specimens to determine whether there is a relation between immunologically mediated renal diseases and the activity of complement receptors that selectively bind antigen-antibody complexes containing activated third component of complement (C3b). These receptors have been termed glomerular complement receptors. Renal lesions associated with in vivo deposition were associated with a loss of receptor sites as demonstrated by reduced or absent in vitro binding of C3b-coated test reagents by glomerular complement receptor. These findings suggest that binding of complement containing immune complexes to glomerular complement receptors in human subjects may participate in the immunopathologic processes of certain immune-complex-mediated renal diseases.

Release of Leukotriene B4 from Sublethally Injured Oligodendrocytes by Terminal Complement Complexes

Abstract: In the present study, the interaction of the terminal complement complexes with Oligodendrocytes was investigated for observation of its effect on membrane lipid hydrolysis. [14C]Arachidonic acid was incorporated into the membrane lipids of cultured Oligodendrocytes before sensitization with anti‐galactocerebroside antiserum. Cells were then exposed to excess C6‐deficient rabbit serum reconstituted with limiting doses of C6 to form various numbers of C5b‐9 complexes. Qualitative analysis of the supernatants by HPLC revealed the presence of compounds that coeluted with arachidonic acid and its oxygenated derivatives, prostaglandin E2, leukotrienes E4 and B4, and 15‐hydroxyeicosatetraenoic acid. The kinetics of leukotriene B4 release by excess C5b‐8 was quantitated by radioimmunoassay. Leukotriene B4 release approached a maximum around 30 min, and C6 dose‐response studies performed at l h showed that maximal levels of leukotriene B4 were detected over a range of sublytic C5b‐9 attack. Maximal release of leukotriene B4 was also achieved by C5b‐8 without further enhancement by addition of lytic doses of C9. Results indicate that sublytic attack of Oligodendrocytes by complement induces release of lipid‐derived inflammatory mediators.

Complement C5 in Experimental Autoimmune Encephalomyelitis (EAE) Facilitates Remyelination and Prevents Gliosis

Activation of the classical complement system is known to play a central role in autoimmune demyelination. We have analyzed the role of complement component C5 in experimental autoimmune encephalomyelitis (EAE) using C5-deficient (C5-d) and C5-sufficient (C5-s) mice. Both groups of mice displayed early onset EAE, a short recovery phase, and similar stable chronic courses. However, in contrast to the clinical similarities, marked differences were apparent by histopathology. During acute EAE in C5-d, a delay in inflammatory cell infiltration and tissue damage was observed along with restricted lesion areas, which in C5-s mice were more extensive and diffuse. More striking were the differences in chronic lesions. In C5-d mice, inflammatory demyelination and Wallerian degeneration were followed by axonal depletion and severe gliosis, while in C5-s, the same initial signs were followed by axonal sparing and extensive remyelination. In C5-d, immunohistochemistry and Western blotting showed an increase in glial fibrillary acidic protein and a decrease in neurofilament protein, proteolipid protein, and several pro-inflammatory markers. These results in the EAE model indicate that absence of C5 resulted in fiber loss and extensive scarring, whereas presence of C5-favored axonal survival and more efficient remyelination.

Induction of IP-10 chemokine promoter by measles virus: comparison with interferon-γ shows the use of the same response element but with differential DNA–protein binding profiles

Measles virus (MV) and interferon (IFN)-gamma induced IP-10 chemokine mRNA in U373 glioblastoma cells. The minimal response element for both MV and IFN-gamma was localized between nucleotide -231 and -153 of muIP-10 promoter, which contains an IFN-stimulated response element (ISRE) and the distal NF-kappa Bd site. Mutation of individual elements showed that ISRE and NF-kappa Bd were required to function together. DNA-protein binding profiles with the minimal response element showed that IFN-gamma induced a complex consisting of STAT1 while MV induced a complex consisting of p50 and p65 in the absence of new protein synthesis. IFN-gamma and MV also induced IRF-1 DNA binding activity which persisted for longer time periods with IFN-gamma stimulation. Despite the functional requirement of both ISRE and NF-kappa Bd elements, different combinations of DNA binding factors are used in the induction of IP-10 by MV or IFN-gamma.

Ultrastructural studies of complement mediated cell death : a biological reaction model to plasma membrane injury

Complement-mediated nucleated cell death has been shown to be independent of colloid-osmotic swelling. In contrast, other factors (e.g. Ca2+ influx) are of importance in the induction of cell death. In this communication, the sequential morphological features of complement-mediated cell injury have been studied by electron microscopy and compared with biochemical data (ATP content and LDH release). It was observed that immediately after C5b-8 lesion formation, although the overall cell morphology is well preserved, the mitochondria display an “ultracondensed” appearance. Upon addition of C9, the mitochondria remain initially condensed, but swell progressively with final formation of flocculent densities. The nuclei become progressively edematous, with concurrent disappearance of heterochromatin. The nucleoli lose their associated chromatin and display segregation of their components with formation of markedly electron-dense filamentous deposits. The nuclear envelope remains initially intact, but subsequently progressive dilatation of the associated perinuclear RER cisterna and distention of the nuclear pores associated with leakage of chromatin into the cytoplasm are seen. The larger cell organelles (including mitochondria, ER, Golgi apparatus, etc.) become clustered around the nucleus, concurrently with marked edema of the outer cytoplasm and bleb formation. The RER cisternae become dilated, whereas the Golgi complex disappears. Relatively early on the plasma membrane shows breaks in continuity. The pattern of these changes — potentially related to Ca2+ influx, ATP efflux and overall metabolic depletion — corresponds to the previously described model of cell reaction to injury, confirming the dynamic nature of the process. The morphology of cell death in this model shares some features, e.g., the nucleolar changes, with “apoptosis” (programmed cell death). However, the overall pattern appears to correspond more to “necrosis,” characterized by loss of volume control and mitochondrial abnormalities.